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Arginyl residues involvement in the microtubule assembly
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 207, No. 2, April 1, pp. 248-255, 1981
Arginyl Residues Involvement in the Microtubule
Assembly
RICARDO B. MACCIONI, JUAN C. VERA, AND JUAN C. SLEBE Instituto de Bioquimica, Facultad de Ciencias, Univemidad
Austral de Chile, Valdivia, Chile
Received April 16, 1980 Modification of pig brain tubulin with 2,3-butanedione, an arginine-specific reagent, resulted in a decrease of its microtubule formation capacity, with apparent first-order kinetics. However, microtubules already assembled were not affected by the reagent. The relation between the polymerization inhibition rate constant and the butanedione concentration followed a saturation curve whereas the colchicine binding activity remained unchanged over that concentration range. GTP partially prevented the decrease of tubulin polymerization induced by the butanedione treatment. This protective effect of GTP was increased by glycerol. The butanedione inhibition of tubulin polymerization appears to be related to the modification of no more than three arginyl residues. These data suggest that at least one of the arginyl residues plays an essential role in tubulin polymerization, probably through its interaction with the negatively charged phosphate moiety of the nucleotide.
The standardization of the conditions for the in vitro reconstitution of microtubules from highly purified tubulin preparations has allowed the study of the assembly mechanism. The requirement of GTP for in vitro tubulin polymerization has been shown in some laboratories (l-3). Affinity labeling studies with different GTP analogs have been performed in order to establish the GTP exchangeable site topography (4, 5). To further understand the chemical nature of the microtubule assembly we have focused our attention on the chemical modification of tubulin’s arginines. There are reasons to believe that arginine residues might be involved in the binding of GTP to tub&n molecules before their polymerization. The role played by arginine residues at the binding site for anionic substrates was recognized after the work by Riordan (6). There is also increasing evidence indicating the participation of the arginine residues at the binding of nucleotide phosphates of many enzymes (7-9). 2,3-Butanedione, a highly selective reagent for the modification of arginyl residues, has been used to produce tubulin 0003-9861/81/040248-08$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
248
modifications. The characteristics of this modification are described in the present report. MATERIAL
AND METHODS
Tubulin from pig brains was prepared after three cycles of polymerization and depolymerization based on the method of Shelanski et al. (10) with modifications. Pig brains were homogenized in 0.5 vol of cold 0.1 M Mes’ (pH 6.4) buffer in 1 M glycerol. The homogenate was centrifuged at 45,OOOg for 60 min at 4°C. The supernatant was adjusted to 1 mM ATP, 0.1 mM GTP, 0.5 mM EGTA, 1 mM MgC&, and 2.5 M glycerol and incubated at 37°C for 30 min. The microtubules thus obtained were spun down at 45,OOOq for 60 min at 30°C. Depolymerization of microtubules was achieved by resuspending the pellet in one-tenth the original volume of 0.1 M Mes buffer (pH 6.4), containing, in addition, 0.5 mM EGTA and 1 mM MgClz, followed by incubation for 15 min at 4°C and centrifugation at 45,OOOgfor 45 min at 4°C. The supernatant was adjusted to 1 mM GTP and 4 M glycerol and a new polymerization was performed as described above. The pellets of microtubules obtained after the second polymerization were immediately frozen in liquid nitrogen and stored at -75’C. 1Abbreviations used: EGTA, ethylene glycol bis(@aminoethyl ether)-hr,W-tetraacetic acid; DTNB, 5,5dithiobis(2-nitrobenzoic acid); MAPS, microtubuleassociated proteins; MES, 2-[N-morpholinolethanesulfonic acid; SDS, sodium dodecyl sulfate.
ARGININES IN MICROTUBULE ASSEMBLY Immediately prior to use, the pellets were resuspended in 0.1 M Mes (pH 6.4) buffer with 0.5 mM EGTA and 1 mM MgCIZ; a depolymerization step was performed as described above followed by the third cycle of polymerization-depolymerization. The last depolymerization step was done in a medium in which MC was omitted. The tubulin solution at a concentration around 10 mg/ml was freed of the remaining unbound nucleotide and the nucleotide bound to the exchangeable site by two extractions with 0.1 ml of a charcsoal suspension containing 1 mM EDTA/ ml of tubulin solution (3). The charcoal suspension was prepared by washing 10 mg of activated charcoal powder with 1 ml of 0.1 M Mes (pH 6.4) containing 2% serum albumin, followed by a wash only in Mes buffer and resuspension in 1 ml of the same buffer solution with 1 mM EDTA. The nucleotide bound to purified tubulin was determined by Iprecipitation of an aliquot of the charcoal-treated ,protein with 5% perchloric acid, centrifugation at 15,000~ for 15 min at 4”C, and measurement. of the absorbance at 255 nm of the supernatant co:ntaining the released guanine nucleotide. On the basis of a molar absorbancy of 12.4 X lo3 M-' cm-’ for (GTP at pH 1.0 (ll), a value of 0.95 mol of residual guanine nucleotide per mole of protein was calculated for the tubulin preparation after the charcoal treatment. Addition of EDTA, as chelating agent for the residual M$+ in the purified tubulin preparation, was shown to be useful in the nucleotide extraction of purified tubulin. Charcoal treatment under these conditions decreased the bound nucleotide from 1.80 to 0.95 mol nucleotide/mol of tubulin dimer. The preparation obtained after the purification procedure described was determined to be 95% tubulin by densitometry of stained polyacrilamide gels electrophoresed in 0.1% SDS. The other 5% represented a minor fraction of microtubule-associated proteins (MAPS). The characteristics of this tubulin preparation have been described elsewhere (12). The polymerization of tubulin was measured by the turbidimetric assay. Thus, changes of the absorbance at 340 nm during the tubulin incubation at 32°C in the presence of 1 mM GTP were recorded. The assay was initiated by the addition of GTP except when tubulin was preincubated at 4°C with the nucleotide. A similar system, but in which GTP was excluded, served as a blank for the polymerization assay. The modification of tubulin with 2,3-butanedione (diacetyl, Sigma Chemical Co.) was performed according to the following steps: Purified tubulin dissolved in 0.1 h1Mes (pH 6.4) buffer, 1 mM Ml?f, and 0.5 mM EGTA was freed of exchangeable GTP by charcoal treatment in the presence of EDTA as described. Fractions of this tubulin preparation were incubated with 50 mM butanedione, unless otherwise stated, at the temperatures indicated; aliquots of the
249
incubation medium were taken at different time intervals and assayed for polymerization with or without addition of GTP. As a control experiment, tubulin samples were incubated under the same conditions mentioned above but without butanedione, and polymerization was assayed as before. In one experiment, butanedione (at a final concentration of 50 mM) was added during the assay, to different tubulin fractions, at various time intervals (Fig. 1). Protein concentrations were determined by the method of Lowry et al. (13) using as standard bovine serum albumin. Electrophoresis in the polyacrylamide-SDS system according to Laemmli (14) was used to check the purity of the tubulin preparations. For amino acid analysis, tubulin was modified with 50 mM butanedione at 25”C, 0.5-ml aliquots were taken at different times, and th reaction was stopped with 0.4 vol of 6 N HCl. The precipitated protein, washed several times, was resuspended in 1 ml of 6 N NC1 and hydrolyzed 24 h at 110°C. Samples of the hydrolysates were taken and analyzed for basic amino acids in a Beckman 120 amino analyzer. Some analyses for the total population of amino acids were also performed. The colchicine binding assay was performed by measuring the radioactivity of [H3]eolchicine (Amersham) bound to tubulin in DE-81 filter disks according to the method of Weisenberg (15). Electron microscope observations of microtubules obtained after polymerization of modified and unmodified tubulin were performed as follows: Aliquots of tubulin samples modified with 50 mM butanedione as described above were obtained at different time intervals and assayed for polymerization. When the polymerization plateau was attained, small fractions (20-30 ~1) were obtained from each polymerization tubulin solution and immediately diluted 1:lO with a solution containing 0.1 M Mes (pH 6.4), 1 mM GTP, 0.5 mM EGTA, 1 M glycerol, and 2% glutaraldehyde. Drops of this fixed preparation were placed on carbon-coated grids, stained with 1% uranyl acetate, and studied under the electron microscope. Aliquots of control samples (unmodified tubulin), obtained at the same time intervals, were polymerized and treated under identical conditions for electron microscopy. RESULTS
In order to study the effect of butanedione on tubulin polymerization, the reagent was added at different stages of the polymerization process (Fig. 1). When butane&one at a concentration of 50 mM was added at time zero, together with GTP, a 40% inhibition of the extent of polymerization was observed. Addition of butaneclione at 5 and 15 min to different tubulin
250
MACCIONI,
VERA,
bation with butanedione increased is shown in Figs. 2A and B. The inactivation rate of tubulin polymerization followed typical pseudo-first-order kinetics. A halflife of 6 min was estimated from the semilogarithmic plot of the decay in polymerization activity. Amino acid composition was analyzed both before and after treatment of tubulin with butanedione for various lengths of time. The data show that a total of three residues of arginine are modified after 40 min of butanedione treatment (Fig. 3). A correlation between the inactivation kinetics of tubulin and the arginine modification is observed in the inset of Fig. 3. Tubulin freed of MAPS by phosphocellulose chromatography was modified under conditions identical to those shown in Fig. 3. When the amino acid compositions of samples taken at different stages of modification were determined, three modified arginine residues were again found. The modification reaction of this tubulin
samples during the polymerization assay failed to produce significant effects in either the extent or the rate of tubulin polymerization, as shown in Fig. 1. Furthermore, additions of the reagent at other time intervals, such as 4, ‘7, and 10 min, did not change significantly the tubulin polymerization as compared with untreated controls. Butanedione at concentrations lower than ‘7 mM did not produce any effects on polymerization kinetics even when added at time zero. After obtaining these results, the effect of butanedione on the free tubulin dimer prior to tubulin polymerization was studied. Incubation of tubulin, freed of exchangeable nucleotide, with the arginine reagent at 25°C in 0.1 M Mes (pH 6.4) buffer resulted in a rapid inactivation of the polymerization of tubulin. This inactivation by butanedione in the presence of Mes buffer was shown to be irreversible. The decrease of both the extent and velocity of polymerization as time of incu-
0
5
AND SLEBE
15
10 TIME
20
25
(min)
FIG. 1. Effect of 2,3-butanedione on tubulin polymerization kinetics. Tubulin (2.6 mg/ml) was incubated in the presence of 1 mM GTP at 32°C and assayed for polymerization (0). Butanedione aliquots were added, to a final concentration of 50 mM, to separated cuvettes containing the polymerization medium at time zero (A), 5 min (a), and 15 min (0) after polymerization had started. The values represent differences in turbidity relative to blanks incubated under the same conditions but without GTP.
ARGININES
IN MICROTUBULE
time of incubation with the modifying agent increased. No microtubules were observed after 40 min of butanedione treatment at 25’C. Thus, the decrease in the number of microtubules dependent on the time of butanedione treatment was related to the inactivation kinetics of tubulin polymerization. Furthermore, microtubules obtained from modified tubulin samples were shorter than those observed in samples from untreated tubulin. A partial loss of the tubular structure in some regions of the microtubule was observed only in those tubules resulting from polymerization of butanedione-treated tubulin. Polymerization kinetics were also stud-
s
‘<
-
20
40
INCUBATION
TIME
60 01
251
ASSEMBLY
Bulonrd,~nr
,
60
100
25’ (mm)
FIG. 2. Effect of incubation time on the inactivation of tubulin polymerization by butanedione. Tubulin at a final concentration of 3 mg/ml was incubated with 50 mM butanedione at 25°C in 0.1 M Mes, pH 6.4, 1 mM Mgz+, 0.5 mM EGTA and 0.7-ml samples were collected at the times indicated and assayed for polymerization as described under Material and Methods (0). In tlhe control experiment, tubulin was incubated in the absence of butanedione and the samples were assayed under the same conditions (0). (A) The percentage remaining polymerization represents the percentage AA340 values (extent of polymerization) with respect to the control at time zero. AAa4,, values are the differences between the absorbance at 340 nm in the plateau of the polymerization kinetics (in the presence of GTP) and the absorbance of the blank (in the absence of GTP). (B) The AA&min values represent the slopes of the polymerization kinetics (polymerization velocity).
freed of MAPS showed kinetic behavior similar to that of tubulin containing MAPS. Neither 1y;syl nor histidyl residues were modified by butanedione, as shown by the amino acid composition. Electron. microscopic observations showed a decrease in the number of microtubules obtained after polymerization of tubulin samples modified with 50 mM butanedione, as compared to those obtained from unmodified samples. The number of :microtubules diminished as the
“0
IO
20 TIME
30
40
(rnln)
FIG. 3. Modification of tubulin arginines with butanedione and inactivation of tubulin polymerization. Tubulin (3 mg/ml) was reacted with 50 mM butanedione as in Fig. 2. Aliquots of 0.5 ml were removed at the times indicated and precipitated with 0.4 vol of 6 N HCl, and the samples were analyzed for amino acid composition. Results were obtained from the differences of the unmodified arginines both before and after butanedione treatment. Different aliquots were obtained at the same time intervals, appropriately diluted, and assayed for polymerization as dsscribed under Material and Methods. The values for modified arginines and those for the residual polymerization activity were obtained as the average of two determinations from different samples of the same tubulin preparation, under identical experimental conditions. The inset shows the decrease of tubulin polymerization activity versus the number of modified arginines.
252
MACCIONI, VERA, AND SLEBE
ied after tubulin treatment at different butanedione concentrations (7-80 mM). A plot of the rate constant for inactivation of tubulin versus butanedione concentration was found not to be linear but rather showed saturation kinetics (Fig. 4). This type of curve suggests that butanedione binds to tubulin arginyl residues, forming an intermediary tubulin-butanedione complex before modification of the residues occurs. A reciprocal plot of the data is shown in the inset of Fig. 4, where an equilibrium constant for the tubulin-butanedione complex of 0.04 M could be calculated. Under the conditions used, Mes buffer does not seem to be a limiting factor, since the rate of inactivation by butanedione was the same for 50 mM and 100 InM Mes. The semilogarithmic plots of the time-dependent inactivation at the different butanedione concentrations was linear for about two half-lives. On the other hand, the colchicine binding activity of tubulin remained unchanged after modification of samples throughout the wide range of butanedione concentrations (Table I).
I 40 [Butanedim.].
80
60
100
mM
FIG. 4. Dependence of the pseudo-first-order rate constant for the inactivation of tubulin on the butanedione concentration. Tubulin at a final concentration of 2.0 mg/ml was incubated at 20°C in 0.1 M Mes (pH 6.4), 1 mM Me, and 0.5 mM EGTA with butanedione at the concentrations indicated in the figure. Aliquots were collected at different times, diluted appropriately, and assayed for polymerization. Pseudo-first-order rate constants were obtained from the logarithmic plot of the kinetic data. The inset shows a reciprocal plot of kob versus butanedione concentration.
TABLE I COLCHICINE BINDING OF TUBULIN TREATED AT DIFFERENT BUTANEDIONE CONCENTRATIONS
Butanedione concentration (rnM)
mol [3H]colchicine/ mol tubulin”
0 7 15 25 40 60 80
0.62 0.63 0.61 0.62 0.61 0.61 0.61
1.00 1.02 0.98 1.00 0.98 0.98 0.98
‘Tubulin samples (100 nl), charcoal treated, in a final concentration of 9.0 mg/ml were incubated at 25’C with butanedione at the concentrations indicated. Aliquots of 10 pl were obtained from each sample after 20 min incubation, diluted 1:lO with 0.1 Mes, and assayed for colchicine binding according to the mzthod of Weisenberg et al. (15). rb = mol [‘H]colchicine/mol tubulin, of butanedione-treated tubuline. r0 = mol [3H]colchicine/mol tubulin, of control (butanedione concentration equals zero).
The inhibition by butanedione of both the extent and rate of tubulin polymerization, as compared with untreated controls, was also measured at different tubulin concentrations ((7,). The extent of polymerization of the modified tubulin was lower than that of the unmodified protein over the whole range of concentrations used, when the butanedione treatment was performed at 4°C under the conditions described in Fig. 5. As a consequence of butanedione treatment the concentration of tubulin necessary to trigger polymerization (critical concentration, C,) increased from 0.35 to 0.65 mg/ml (Fig. 5). In turn, this increase implies a decrease in the association constant (K,) for the addition of tubulin dimer to the growing microtubule, according to the model proposed by Oosawa and Higashi (16). In addition to the increase of C,, the slope of the line resulting from the plot of polymerization versus tubulin concentration was lower in the modified samples than in the controls. The modification was also performed in the presence of GTP. Pretreatment of tu-
253
ARGININES IN MICROTUBULE ASSEMBLY
seems to be responsible for this assembly inhibition. Arginine has been considered as an essential residue in many enzymes and it has been thought to interact with the negatively charged phosphate group of the substrate (8, 9). As suggested by Lange et al. (19), arginine residues play a role as a positively charged center for the interaction of anionic ligands. The inhibition of tubulin polymerization induced by butanedione was not due to protein denaturation as demonstrated by the slight inactivation of the unmodified tubulin (controls) as compared with the modified protein. TUBULIN,
mglml
FIG. 5. Effect of tubulin concentration on the inactivation by butanedione. Tubulin samples at the concentrations indicated were incubated at 4’C for 20 min with !50mM butanedione. Aliquots were then obtained and1the polymerization was assayed (0). The control samples were incubated under the same conditions but without butanedione (0). AA%0values are the differences between the absorbance at 340 nm in the plateau of the polymerization assay and the absorbance of the blank, as indicated in Fig. 2.
bulin with GTP partially prevented the inhibition of tubulin polymerization induced by butanedione treatment, thus suggesting a protective effect of GTP (Fig. 6A). This protective effect of the tubulin polymerization was enhanced when the preincubation was performed in the presence of 1 M glycerol prior to the butanedione modification (Fig. 6B).
B
0
DISCUSSION
Despite increasing interest in the study of the tubulin polymerization mechanism, little is known about the functional role of specific residues of the protein. Previous reports have implicated histidine (1’7) and sulfhydrylic groups (18) in tubulin assembly. The present report describes the results of the chemical modification of neurotubulin with butanedione. The treatment of tubulin with butanedione inhibits both the extent and rate of self-assembly of the dimer to form microtubules. Modification of arginine residues
40 INCUBATION
SO
120
160
TIME at Lo fmin)
FIG. 6. Effect of GTP on the inactivation of tubulin by butanedione. (A) Tubulin (3 mg/ml) was incubated at 4°C with 50 mM butanedione and aliquots were taken at different times and assayed for polymerization (0). Tubulin was first preincubated at 4°C for 10 min with 2 mM GTP, and then butanedione was added, at time zero, to the incubation medium. Aliquots were collected at the time intervals indicated in the figure and assayed for polymerization (A). Control samples were incubated in the absence of both, GTP and butanedione, and the aliquots were assayed for polymerization (0). (B) Samples were treated under the same conditions given in (A) except that 1 M glycerol was added.
254
MACCIONI,
VERA,
Other residues of tubulin did not appear to be affected by butanedione treatment under the experimental conditions described. Amino acid analysis of the modified tubulin indicates that histidyl and lysyl residues remained unchanged after butanedione treatment. Sulfhydrylic groups, which have been shown to be important in tubulin polymerization, were not modified by butanedione either, since the DTNB titration study revealed an equivalent number of cysteines in both modified and unmodified tubulins. Three arginyl residues were modified in tubulin samples in the presence or in the absence of a minor fraction of MAPS. This finding and the similarities in the reactivity of the residues from tubulin and tubulin-MAPS, as indicated by the modification kinetics, suggest that the arginyl residues related to the tubulin inactivation by butanedione are located in the tubulin molecule. Although three arginine residues were modified by treatment of tubulin with butanedione, the relation of residual polymerization versus the number of modified arginines (inset, Fig. 3) suggests that at least one residue could be essential for polymerization. Tubulin polymerization was increasingly inhibited by increasing concentrations of 2,3-butanedione. The saturation curve observed for the relation between kobsand butanedione concentration (Fig. 4) suggests that butanedione binds to tubulin before the modification of arginyl residues takes place. This type of saturation function was also observed in the butanedione modification of alcohol dehydrogenase (19). However, butanedione inactivation of tubulin appears to be different from butanedione inactivation of some enzymes. Diphosphopyridine nucleotide-isocitrate dehydrogenase (20) and mitochondrial adenosine triphosphatase (9) are examples of enzymes with linearity between kobsand butanedione concentration. It was of interest to observe if the modifying reagent had any effect on the colchicine binding capacity. Colchicine binding remained unchanged after the tubulin treatment with different butanedione con-
AND SLEBE
centrations, even under strong modification conditions. This result suggests that arginine residues are probably not involved in colchicine binding to the tubulin molecule. Protection experiments showed that GTP partially prevented the effects produced by butanedione (Fig. 6A). This finding and that referring to the modification of arginine by butanedione suggest that there is at least one arginine residue closely related to the GTP binding site that might be involved in the polymerization process. As in other systems (7-g), a likely explanation for the role of the arginine residue in tubulin assembly is that it interacts with the negatively charged phosphate of the nucleotide, as a prelude to the assembly process. An alternative interpretation of the data of the GTP protection experiment should be that an arginyl residue, sensitive to modification in the unprotected enzyme, could be masked after a conformational change of tubulin induced by the nucleotide. Glycerol has been shown to enhance microtubule formation through general thermodynamic interaction with tubulin (21). The glycerol enhancement of the protective effect of GTP, on one hand, and the mild protection by glycerol (Fig. 6B) against the loss of tubulin polymerization induced by butanedione, on the other, could be best explained if there is a nonspecific interaction between glycerol and the protein. The results presented in this study show that three arginyl residues of tubulin are modified by butanedione treatment and indicate that at least one of them is involved in the tubulin assembly into microtubules. ACKNOWLEDGMENTS We gratefully acknowledge the helpful comments of Dr. Esteban Rodriguez. We thank Mrs. Hella Ludwig and Miss Monica Brito for technical assistance, Mr. Luis Delannoy for assistance with the electron microscope, and Mrs. Isabel Romero and Maria A. Espinoza for collaboration in the preparation of the manuscript. This investigation was supported by
ARGININES Research Grant the Universidad
S-79-9 from the Research Austral de Chile.
IN MICROTUBULE Fund of
REFERENCES 1. GASKIN, F., CANTOR, C. R.. AND SHELANSKI, M. L. (1974) J. MoL BioL 89. 737-758. 2. OLMSTED, J. B., AND BORISY, G. G. (1975) Biu
chemistry 14, 2996-3005. 3. MACCIONI, R., ASD SEEDS, N. W. (1977) Proc. Nat. Acad. Sci. USA 74, 462-466. 4. GEAHLEN, R. L., AND HALEY, B. E. (1977) Proc. Nat. Acad Sci. USA 74.4375-4377. 5. MACCIONI, R., AND SEEDS, N. W. (1977) J. CeU. Biol. 75. 285a. 6. RIORDAN, J. F. (1973) Biochemistry 12.3915-3923. 7. MARCUS, F. (1975) Biochemistry 14,3916-3921. 8. BORDERS, C. L., AND RIORDAS, J. F. (1975) Biochemistry 14, 4699-4704. 9. MARCUS, F., SCHUSTER, S. M., ANDLARDY, H. A. (1976) .J. BioL Chem. 251, 1775-1780. 10. SHELANSKI, M. L., GASKIN, F., AND CANTOR, C. R. (1973) Proc. Nat. Acad Sci USA 70.765-
768. 11. ARAI, T., IIIARA, Y., ARAI, K., AND KAZIRO, Y.
(1975) .I. B&hem.
77, 647-658.
ASSEMBLY
255
12. MACCIONI, R. B., VERA, J. C., AND SLEBE, J. C. (1980) in Gene and Protein Behavior (Weisabath, H., Siddiqui, M. A. Q., and Krauskopf, M., eds.), Academic Press, New York. 13. LOWRY. 0. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193.
265-275. 14. LAEMMLI, U. K. (1970) Nature &dun)
222,680-
685. 15. WEISENBERG, R. C., B~RISY. G. G., AND TAWR, E. W. (1968) Biochemistry 7,4466-4479. 16. OOSAWA. F., ANDHIGASHI. S. (1967) Progr. Theor. BioL 1, 79-164. 17. LEE, Y. C., HOUSTON, L. L., AND HIMES, R. H. (1976) Biochem. Biophys. Res. Commun 70,
50-57. 18. NISHIDA, E., AND KOBAYASHI, T. (1977) J. B&hem
81,343-347. 19. LANGE, L. G., RIORDAN, J. F., AND VALLEE, B. L.
(1974) Biochemistry 13.4361-4370. 20. HAYMAN, S., AND COLMAN, R. F. (1978) Biochemistry 17,4161-4168. 21. LEE, J. C., ANDTIMASHEFF, S. N. (1977) Biochemistry 16.1754-1763.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 207, No. 2, April 1, pp. 248-255, 1981
Arginyl Residues Involvement in the Microtubule
Assembly
RICARDO B. MACCIONI, JUAN C. VERA, AND JUAN C. SLEBE Instituto de Bioquimica, Facultad de Ciencias, Univemidad
Austral de Chile, Valdivia, Chile
Received April 16, 1980 Modification of pig brain tubulin with 2,3-butanedione, an arginine-specific reagent, resulted in a decrease of its microtubule formation capacity, with apparent first-order kinetics. However, microtubules already assembled were not affected by the reagent. The relation between the polymerization inhibition rate constant and the butanedione concentration followed a saturation curve whereas the colchicine binding activity remained unchanged over that concentration range. GTP partially prevented the decrease of tubulin polymerization induced by the butanedione treatment. This protective effect of GTP was increased by glycerol. The butanedione inhibition of tubulin polymerization appears to be related to the modification of no more than three arginyl residues. These data suggest that at least one of the arginyl residues plays an essential role in tubulin polymerization, probably through its interaction with the negatively charged phosphate moiety of the nucleotide.
The standardization of the conditions for the in vitro reconstitution of microtubules from highly purified tubulin preparations has allowed the study of the assembly mechanism. The requirement of GTP for in vitro tubulin polymerization has been shown in some laboratories (l-3). Affinity labeling studies with different GTP analogs have been performed in order to establish the GTP exchangeable site topography (4, 5). To further understand the chemical nature of the microtubule assembly we have focused our attention on the chemical modification of tubulin’s arginines. There are reasons to believe that arginine residues might be involved in the binding of GTP to tub&n molecules before their polymerization. The role played by arginine residues at the binding site for anionic substrates was recognized after the work by Riordan (6). There is also increasing evidence indicating the participation of the arginine residues at the binding of nucleotide phosphates of many enzymes (7-9). 2,3-Butanedione, a highly selective reagent for the modification of arginyl residues, has been used to produce tubulin 0003-9861/81/040248-08$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
248
modifications. The characteristics of this modification are described in the present report. MATERIAL
AND METHODS
Tubulin from pig brains was prepared after three cycles of polymerization and depolymerization based on the method of Shelanski et al. (10) with modifications. Pig brains were homogenized in 0.5 vol of cold 0.1 M Mes’ (pH 6.4) buffer in 1 M glycerol. The homogenate was centrifuged at 45,OOOg for 60 min at 4°C. The supernatant was adjusted to 1 mM ATP, 0.1 mM GTP, 0.5 mM EGTA, 1 mM MgC&, and 2.5 M glycerol and incubated at 37°C for 30 min. The microtubules thus obtained were spun down at 45,OOOq for 60 min at 30°C. Depolymerization of microtubules was achieved by resuspending the pellet in one-tenth the original volume of 0.1 M Mes buffer (pH 6.4), containing, in addition, 0.5 mM EGTA and 1 mM MgClz, followed by incubation for 15 min at 4°C and centrifugation at 45,OOOgfor 45 min at 4°C. The supernatant was adjusted to 1 mM GTP and 4 M glycerol and a new polymerization was performed as described above. The pellets of microtubules obtained after the second polymerization were immediately frozen in liquid nitrogen and stored at -75’C. 1Abbreviations used: EGTA, ethylene glycol bis(@aminoethyl ether)-hr,W-tetraacetic acid; DTNB, 5,5dithiobis(2-nitrobenzoic acid); MAPS, microtubuleassociated proteins; MES, 2-[N-morpholinolethanesulfonic acid; SDS, sodium dodecyl sulfate.
ARGININES IN MICROTUBULE ASSEMBLY Immediately prior to use, the pellets were resuspended in 0.1 M Mes (pH 6.4) buffer with 0.5 mM EGTA and 1 mM MgCIZ; a depolymerization step was performed as described above followed by the third cycle of polymerization-depolymerization. The last depolymerization step was done in a medium in which MC was omitted. The tubulin solution at a concentration around 10 mg/ml was freed of the remaining unbound nucleotide and the nucleotide bound to the exchangeable site by two extractions with 0.1 ml of a charcsoal suspension containing 1 mM EDTA/ ml of tubulin solution (3). The charcoal suspension was prepared by washing 10 mg of activated charcoal powder with 1 ml of 0.1 M Mes (pH 6.4) containing 2% serum albumin, followed by a wash only in Mes buffer and resuspension in 1 ml of the same buffer solution with 1 mM EDTA. The nucleotide bound to purified tubulin was determined by Iprecipitation of an aliquot of the charcoal-treated ,protein with 5% perchloric acid, centrifugation at 15,000~ for 15 min at 4”C, and measurement. of the absorbance at 255 nm of the supernatant co:ntaining the released guanine nucleotide. On the basis of a molar absorbancy of 12.4 X lo3 M-' cm-’ for (GTP at pH 1.0 (ll), a value of 0.95 mol of residual guanine nucleotide per mole of protein was calculated for the tubulin preparation after the charcoal treatment. Addition of EDTA, as chelating agent for the residual M$+ in the purified tubulin preparation, was shown to be useful in the nucleotide extraction of purified tubulin. Charcoal treatment under these conditions decreased the bound nucleotide from 1.80 to 0.95 mol nucleotide/mol of tubulin dimer. The preparation obtained after the purification procedure described was determined to be 95% tubulin by densitometry of stained polyacrilamide gels electrophoresed in 0.1% SDS. The other 5% represented a minor fraction of microtubule-associated proteins (MAPS). The characteristics of this tubulin preparation have been described elsewhere (12). The polymerization of tubulin was measured by the turbidimetric assay. Thus, changes of the absorbance at 340 nm during the tubulin incubation at 32°C in the presence of 1 mM GTP were recorded. The assay was initiated by the addition of GTP except when tubulin was preincubated at 4°C with the nucleotide. A similar system, but in which GTP was excluded, served as a blank for the polymerization assay. The modification of tubulin with 2,3-butanedione (diacetyl, Sigma Chemical Co.) was performed according to the following steps: Purified tubulin dissolved in 0.1 h1Mes (pH 6.4) buffer, 1 mM Ml?f, and 0.5 mM EGTA was freed of exchangeable GTP by charcoal treatment in the presence of EDTA as described. Fractions of this tubulin preparation were incubated with 50 mM butanedione, unless otherwise stated, at the temperatures indicated; aliquots of the
249
incubation medium were taken at different time intervals and assayed for polymerization with or without addition of GTP. As a control experiment, tubulin samples were incubated under the same conditions mentioned above but without butanedione, and polymerization was assayed as before. In one experiment, butanedione (at a final concentration of 50 mM) was added during the assay, to different tubulin fractions, at various time intervals (Fig. 1). Protein concentrations were determined by the method of Lowry et al. (13) using as standard bovine serum albumin. Electrophoresis in the polyacrylamide-SDS system according to Laemmli (14) was used to check the purity of the tubulin preparations. For amino acid analysis, tubulin was modified with 50 mM butanedione at 25”C, 0.5-ml aliquots were taken at different times, and th reaction was stopped with 0.4 vol of 6 N HCl. The precipitated protein, washed several times, was resuspended in 1 ml of 6 N NC1 and hydrolyzed 24 h at 110°C. Samples of the hydrolysates were taken and analyzed for basic amino acids in a Beckman 120 amino analyzer. Some analyses for the total population of amino acids were also performed. The colchicine binding assay was performed by measuring the radioactivity of [H3]eolchicine (Amersham) bound to tubulin in DE-81 filter disks according to the method of Weisenberg (15). Electron microscope observations of microtubules obtained after polymerization of modified and unmodified tubulin were performed as follows: Aliquots of tubulin samples modified with 50 mM butanedione as described above were obtained at different time intervals and assayed for polymerization. When the polymerization plateau was attained, small fractions (20-30 ~1) were obtained from each polymerization tubulin solution and immediately diluted 1:lO with a solution containing 0.1 M Mes (pH 6.4), 1 mM GTP, 0.5 mM EGTA, 1 M glycerol, and 2% glutaraldehyde. Drops of this fixed preparation were placed on carbon-coated grids, stained with 1% uranyl acetate, and studied under the electron microscope. Aliquots of control samples (unmodified tubulin), obtained at the same time intervals, were polymerized and treated under identical conditions for electron microscopy. RESULTS
In order to study the effect of butanedione on tubulin polymerization, the reagent was added at different stages of the polymerization process (Fig. 1). When butane&one at a concentration of 50 mM was added at time zero, together with GTP, a 40% inhibition of the extent of polymerization was observed. Addition of butaneclione at 5 and 15 min to different tubulin
250
MACCIONI,
VERA,
bation with butanedione increased is shown in Figs. 2A and B. The inactivation rate of tubulin polymerization followed typical pseudo-first-order kinetics. A halflife of 6 min was estimated from the semilogarithmic plot of the decay in polymerization activity. Amino acid composition was analyzed both before and after treatment of tubulin with butanedione for various lengths of time. The data show that a total of three residues of arginine are modified after 40 min of butanedione treatment (Fig. 3). A correlation between the inactivation kinetics of tubulin and the arginine modification is observed in the inset of Fig. 3. Tubulin freed of MAPS by phosphocellulose chromatography was modified under conditions identical to those shown in Fig. 3. When the amino acid compositions of samples taken at different stages of modification were determined, three modified arginine residues were again found. The modification reaction of this tubulin
samples during the polymerization assay failed to produce significant effects in either the extent or the rate of tubulin polymerization, as shown in Fig. 1. Furthermore, additions of the reagent at other time intervals, such as 4, ‘7, and 10 min, did not change significantly the tubulin polymerization as compared with untreated controls. Butanedione at concentrations lower than ‘7 mM did not produce any effects on polymerization kinetics even when added at time zero. After obtaining these results, the effect of butanedione on the free tubulin dimer prior to tubulin polymerization was studied. Incubation of tubulin, freed of exchangeable nucleotide, with the arginine reagent at 25°C in 0.1 M Mes (pH 6.4) buffer resulted in a rapid inactivation of the polymerization of tubulin. This inactivation by butanedione in the presence of Mes buffer was shown to be irreversible. The decrease of both the extent and velocity of polymerization as time of incu-
0
5
AND SLEBE
15
10 TIME
20
25
(min)
FIG. 1. Effect of 2,3-butanedione on tubulin polymerization kinetics. Tubulin (2.6 mg/ml) was incubated in the presence of 1 mM GTP at 32°C and assayed for polymerization (0). Butanedione aliquots were added, to a final concentration of 50 mM, to separated cuvettes containing the polymerization medium at time zero (A), 5 min (a), and 15 min (0) after polymerization had started. The values represent differences in turbidity relative to blanks incubated under the same conditions but without GTP.
ARGININES
IN MICROTUBULE
time of incubation with the modifying agent increased. No microtubules were observed after 40 min of butanedione treatment at 25’C. Thus, the decrease in the number of microtubules dependent on the time of butanedione treatment was related to the inactivation kinetics of tubulin polymerization. Furthermore, microtubules obtained from modified tubulin samples were shorter than those observed in samples from untreated tubulin. A partial loss of the tubular structure in some regions of the microtubule was observed only in those tubules resulting from polymerization of butanedione-treated tubulin. Polymerization kinetics were also stud-
s
‘<
-
20
40
INCUBATION
TIME
60 01
251
ASSEMBLY
Bulonrd,~nr
,
60
100
25’ (mm)
FIG. 2. Effect of incubation time on the inactivation of tubulin polymerization by butanedione. Tubulin at a final concentration of 3 mg/ml was incubated with 50 mM butanedione at 25°C in 0.1 M Mes, pH 6.4, 1 mM Mgz+, 0.5 mM EGTA and 0.7-ml samples were collected at the times indicated and assayed for polymerization as described under Material and Methods (0). In tlhe control experiment, tubulin was incubated in the absence of butanedione and the samples were assayed under the same conditions (0). (A) The percentage remaining polymerization represents the percentage AA340 values (extent of polymerization) with respect to the control at time zero. AAa4,, values are the differences between the absorbance at 340 nm in the plateau of the polymerization kinetics (in the presence of GTP) and the absorbance of the blank (in the absence of GTP). (B) The AA&min values represent the slopes of the polymerization kinetics (polymerization velocity).
freed of MAPS showed kinetic behavior similar to that of tubulin containing MAPS. Neither 1y;syl nor histidyl residues were modified by butanedione, as shown by the amino acid composition. Electron. microscopic observations showed a decrease in the number of microtubules obtained after polymerization of tubulin samples modified with 50 mM butanedione, as compared to those obtained from unmodified samples. The number of :microtubules diminished as the
“0
IO
20 TIME
30
40
(rnln)
FIG. 3. Modification of tubulin arginines with butanedione and inactivation of tubulin polymerization. Tubulin (3 mg/ml) was reacted with 50 mM butanedione as in Fig. 2. Aliquots of 0.5 ml were removed at the times indicated and precipitated with 0.4 vol of 6 N HCl, and the samples were analyzed for amino acid composition. Results were obtained from the differences of the unmodified arginines both before and after butanedione treatment. Different aliquots were obtained at the same time intervals, appropriately diluted, and assayed for polymerization as dsscribed under Material and Methods. The values for modified arginines and those for the residual polymerization activity were obtained as the average of two determinations from different samples of the same tubulin preparation, under identical experimental conditions. The inset shows the decrease of tubulin polymerization activity versus the number of modified arginines.
252
MACCIONI, VERA, AND SLEBE
ied after tubulin treatment at different butanedione concentrations (7-80 mM). A plot of the rate constant for inactivation of tubulin versus butanedione concentration was found not to be linear but rather showed saturation kinetics (Fig. 4). This type of curve suggests that butanedione binds to tubulin arginyl residues, forming an intermediary tubulin-butanedione complex before modification of the residues occurs. A reciprocal plot of the data is shown in the inset of Fig. 4, where an equilibrium constant for the tubulin-butanedione complex of 0.04 M could be calculated. Under the conditions used, Mes buffer does not seem to be a limiting factor, since the rate of inactivation by butanedione was the same for 50 mM and 100 InM Mes. The semilogarithmic plots of the time-dependent inactivation at the different butanedione concentrations was linear for about two half-lives. On the other hand, the colchicine binding activity of tubulin remained unchanged after modification of samples throughout the wide range of butanedione concentrations (Table I).
I 40 [Butanedim.].
80
60
100
mM
FIG. 4. Dependence of the pseudo-first-order rate constant for the inactivation of tubulin on the butanedione concentration. Tubulin at a final concentration of 2.0 mg/ml was incubated at 20°C in 0.1 M Mes (pH 6.4), 1 mM Me, and 0.5 mM EGTA with butanedione at the concentrations indicated in the figure. Aliquots were collected at different times, diluted appropriately, and assayed for polymerization. Pseudo-first-order rate constants were obtained from the logarithmic plot of the kinetic data. The inset shows a reciprocal plot of kob versus butanedione concentration.
TABLE I COLCHICINE BINDING OF TUBULIN TREATED AT DIFFERENT BUTANEDIONE CONCENTRATIONS
Butanedione concentration (rnM)
mol [3H]colchicine/ mol tubulin”
0 7 15 25 40 60 80
0.62 0.63 0.61 0.62 0.61 0.61 0.61
1.00 1.02 0.98 1.00 0.98 0.98 0.98
‘Tubulin samples (100 nl), charcoal treated, in a final concentration of 9.0 mg/ml were incubated at 25’C with butanedione at the concentrations indicated. Aliquots of 10 pl were obtained from each sample after 20 min incubation, diluted 1:lO with 0.1 Mes, and assayed for colchicine binding according to the mzthod of Weisenberg et al. (15). rb = mol [‘H]colchicine/mol tubulin, of butanedione-treated tubuline. r0 = mol [3H]colchicine/mol tubulin, of control (butanedione concentration equals zero).
The inhibition by butanedione of both the extent and rate of tubulin polymerization, as compared with untreated controls, was also measured at different tubulin concentrations ((7,). The extent of polymerization of the modified tubulin was lower than that of the unmodified protein over the whole range of concentrations used, when the butanedione treatment was performed at 4°C under the conditions described in Fig. 5. As a consequence of butanedione treatment the concentration of tubulin necessary to trigger polymerization (critical concentration, C,) increased from 0.35 to 0.65 mg/ml (Fig. 5). In turn, this increase implies a decrease in the association constant (K,) for the addition of tubulin dimer to the growing microtubule, according to the model proposed by Oosawa and Higashi (16). In addition to the increase of C,, the slope of the line resulting from the plot of polymerization versus tubulin concentration was lower in the modified samples than in the controls. The modification was also performed in the presence of GTP. Pretreatment of tu-
253
ARGININES IN MICROTUBULE ASSEMBLY
seems to be responsible for this assembly inhibition. Arginine has been considered as an essential residue in many enzymes and it has been thought to interact with the negatively charged phosphate group of the substrate (8, 9). As suggested by Lange et al. (19), arginine residues play a role as a positively charged center for the interaction of anionic ligands. The inhibition of tubulin polymerization induced by butanedione was not due to protein denaturation as demonstrated by the slight inactivation of the unmodified tubulin (controls) as compared with the modified protein. TUBULIN,
mglml
FIG. 5. Effect of tubulin concentration on the inactivation by butanedione. Tubulin samples at the concentrations indicated were incubated at 4’C for 20 min with !50mM butanedione. Aliquots were then obtained and1the polymerization was assayed (0). The control samples were incubated under the same conditions but without butanedione (0). AA%0values are the differences between the absorbance at 340 nm in the plateau of the polymerization assay and the absorbance of the blank, as indicated in Fig. 2.
bulin with GTP partially prevented the inhibition of tubulin polymerization induced by butanedione treatment, thus suggesting a protective effect of GTP (Fig. 6A). This protective effect of the tubulin polymerization was enhanced when the preincubation was performed in the presence of 1 M glycerol prior to the butanedione modification (Fig. 6B).
B
0
DISCUSSION
Despite increasing interest in the study of the tubulin polymerization mechanism, little is known about the functional role of specific residues of the protein. Previous reports have implicated histidine (1’7) and sulfhydrylic groups (18) in tubulin assembly. The present report describes the results of the chemical modification of neurotubulin with butanedione. The treatment of tubulin with butanedione inhibits both the extent and rate of self-assembly of the dimer to form microtubules. Modification of arginine residues
40 INCUBATION
SO
120
160
TIME at Lo fmin)
FIG. 6. Effect of GTP on the inactivation of tubulin by butanedione. (A) Tubulin (3 mg/ml) was incubated at 4°C with 50 mM butanedione and aliquots were taken at different times and assayed for polymerization (0). Tubulin was first preincubated at 4°C for 10 min with 2 mM GTP, and then butanedione was added, at time zero, to the incubation medium. Aliquots were collected at the time intervals indicated in the figure and assayed for polymerization (A). Control samples were incubated in the absence of both, GTP and butanedione, and the aliquots were assayed for polymerization (0). (B) Samples were treated under the same conditions given in (A) except that 1 M glycerol was added.
254
MACCIONI,
VERA,
Other residues of tubulin did not appear to be affected by butanedione treatment under the experimental conditions described. Amino acid analysis of the modified tubulin indicates that histidyl and lysyl residues remained unchanged after butanedione treatment. Sulfhydrylic groups, which have been shown to be important in tubulin polymerization, were not modified by butanedione either, since the DTNB titration study revealed an equivalent number of cysteines in both modified and unmodified tubulins. Three arginyl residues were modified in tubulin samples in the presence or in the absence of a minor fraction of MAPS. This finding and the similarities in the reactivity of the residues from tubulin and tubulin-MAPS, as indicated by the modification kinetics, suggest that the arginyl residues related to the tubulin inactivation by butanedione are located in the tubulin molecule. Although three arginine residues were modified by treatment of tubulin with butanedione, the relation of residual polymerization versus the number of modified arginines (inset, Fig. 3) suggests that at least one residue could be essential for polymerization. Tubulin polymerization was increasingly inhibited by increasing concentrations of 2,3-butanedione. The saturation curve observed for the relation between kobsand butanedione concentration (Fig. 4) suggests that butanedione binds to tubulin before the modification of arginyl residues takes place. This type of saturation function was also observed in the butanedione modification of alcohol dehydrogenase (19). However, butanedione inactivation of tubulin appears to be different from butanedione inactivation of some enzymes. Diphosphopyridine nucleotide-isocitrate dehydrogenase (20) and mitochondrial adenosine triphosphatase (9) are examples of enzymes with linearity between kobsand butanedione concentration. It was of interest to observe if the modifying reagent had any effect on the colchicine binding capacity. Colchicine binding remained unchanged after the tubulin treatment with different butanedione con-
AND SLEBE
centrations, even under strong modification conditions. This result suggests that arginine residues are probably not involved in colchicine binding to the tubulin molecule. Protection experiments showed that GTP partially prevented the effects produced by butanedione (Fig. 6A). This finding and that referring to the modification of arginine by butanedione suggest that there is at least one arginine residue closely related to the GTP binding site that might be involved in the polymerization process. As in other systems (7-g), a likely explanation for the role of the arginine residue in tubulin assembly is that it interacts with the negatively charged phosphate of the nucleotide, as a prelude to the assembly process. An alternative interpretation of the data of the GTP protection experiment should be that an arginyl residue, sensitive to modification in the unprotected enzyme, could be masked after a conformational change of tubulin induced by the nucleotide. Glycerol has been shown to enhance microtubule formation through general thermodynamic interaction with tubulin (21). The glycerol enhancement of the protective effect of GTP, on one hand, and the mild protection by glycerol (Fig. 6B) against the loss of tubulin polymerization induced by butanedione, on the other, could be best explained if there is a nonspecific interaction between glycerol and the protein. The results presented in this study show that three arginyl residues of tubulin are modified by butanedione treatment and indicate that at least one of them is involved in the tubulin assembly into microtubules. ACKNOWLEDGMENTS We gratefully acknowledge the helpful comments of Dr. Esteban Rodriguez. We thank Mrs. Hella Ludwig and Miss Monica Brito for technical assistance, Mr. Luis Delannoy for assistance with the electron microscope, and Mrs. Isabel Romero and Maria A. Espinoza for collaboration in the preparation of the manuscript. This investigation was supported by
ARGININES Research Grant the Universidad
S-79-9 from the Research Austral de Chile.
IN MICROTUBULE Fund of
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chemistry 14, 2996-3005. 3. MACCIONI, R., ASD SEEDS, N. W. (1977) Proc. Nat. Acad. Sci. USA 74, 462-466. 4. GEAHLEN, R. L., AND HALEY, B. E. (1977) Proc. Nat. Acad Sci. USA 74.4375-4377. 5. MACCIONI, R., AND SEEDS, N. W. (1977) J. CeU. Biol. 75. 285a. 6. RIORDAN, J. F. (1973) Biochemistry 12.3915-3923. 7. MARCUS, F. (1975) Biochemistry 14,3916-3921. 8. BORDERS, C. L., AND RIORDAS, J. F. (1975) Biochemistry 14, 4699-4704. 9. MARCUS, F., SCHUSTER, S. M., ANDLARDY, H. A. (1976) .J. BioL Chem. 251, 1775-1780. 10. SHELANSKI, M. L., GASKIN, F., AND CANTOR, C. R. (1973) Proc. Nat. Acad Sci USA 70.765-
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ASSEMBLY
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12. MACCIONI, R. B., VERA, J. C., AND SLEBE, J. C. (1980) in Gene and Protein Behavior (Weisabath, H., Siddiqui, M. A. Q., and Krauskopf, M., eds.), Academic Press, New York. 13. LOWRY. 0. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193.
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685. 15. WEISENBERG, R. C., B~RISY. G. G., AND TAWR, E. W. (1968) Biochemistry 7,4466-4479. 16. OOSAWA. F., ANDHIGASHI. S. (1967) Progr. Theor. BioL 1, 79-164. 17. LEE, Y. C., HOUSTON, L. L., AND HIMES, R. H. (1976) Biochem. Biophys. Res. Commun 70,
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